The present invention relates to nucleotide sequences that encode the complementarity-determining regions (CDRs) and framework regions (FR) of antibodies. The invention particularly relates to CDRs and FRs of anti-idiotypic antibodies that recognize the idiotopes of anti-HIV antibodies. The nucleotide sequences are pertinent to modulation of the immune response to HIV in HIV-1 infected individuals, as by therapeutic vaccination with the anti-idiotypic antibodies or antibody fragments they encode, as well as by direct therapeutic administration as DNA molecules.
Acquired Immune Deficiency Syndrome (AIDS) has claimed the lives of millions of people worldwide and continues to be a leading cause of death, particularly in under-developed countries. The primary etiologic agent of AIDS is widely accepted to be one or more strains of the human immunodeficiency virus (HIV). The most studied strain of HIV is the type 1 strain (HIV-1), which is also referred to as HTLV-III. An intact HIV-1 virion is roughly spherical and presents an outer glycoprotein membrane covered with distinctive knobs and spikes.
Following initial infection with HIV is an asymptomatic stage during which the host harbors the virus and tests seropositive for HIV-1 antibodies. This stage can last as long as five years or more. This stage is followed by an AIDS-related complex stage (ARC) and, finally, AIDS. The final stage of AIDS is characterized by a variety of opportunistic infections due to the reduced vitality of the immune system. Although several drugs and drug combinations have been shown to alleviate the symptoms of AIDS and evidently reduce the viral load, to date, no effective prophylactic or therapeutic vaccine against HIV infection has been approved.
One approach proposed for the development of novel therapeutic antibodies or therapeutic vaccines, as well as prophylactic co-vaccines, employs so-called anti-idiotypic antibodies, and fragments thereof. A second strategy for modulation of immune response against viral infection involves direct inoculation of tissues in vivo with DNA encoding the VH and/or VL chains of an anti-idiotypic antibody as a therapeutic DNA vaccine.
Previously, an anti-idiotypic antibody has been shown to induce immune modulation in HIV infection. A murine anti-idiotypic antibody (designated 1F7 and produced by hybridoma ATCC Accession No. HB 11286) has been raised against pooled human anti-HIV-1 antibodies. [Mueller, S., et al., J. Immunol. (1991), 147:933-941]. The 1F7 idiotype is shared by human anti-HIV-1 antibodies having specificity for different proteins (envelope, core, and reverse transcriptase) of HIV-1, and the idiotype is shared by more than 70% of HIV-infected individuals. [Wang, H. et al., Eur. J. Immunol. (1992), 22:1749-1755]. The 1F7 idiotype occurs in high levels among patients with HIV lymphoma; however, this is shown to be due to HIV infection rather than B-cell neoplasia or abnormal B-cell proliferation. [Herndier, B., et al., Hybridoma (1993), 12: 529-537]. Findings suggest that the 1F7 idiotype is a marker for B-cell clones induced during the primary immune response to HIV and maintained throughout life. [Mueller, S., et al., Hybridoma (1997), 16: 17-21].
The potential for Mab 1F7 as a therapeutic antibody (therapeutic vaccine) has recently been demonstrated in rhesus macaques infected with a simian variant of HIV (SHIV-IIIB). [S. Mueller, et al., PNAS (1998) 95: 276-281; U.S. Pat. No. 6,057,421]. In these studies, a series of intravenous injections of purified 1F7 antibody enhanced and broadened the macaques"" virus neutralizing antibody response to the simian virus. This result suggests the potential of beneficially modulating the immune response of chronically HIV-1 infected individuals without clinical side effects in addition to, or during breaks in, antiviral drug therapy.
The functionality of the anti-idiotypic antibody 1F7 may be exploited directly according to the following approaches:
(1) 1F7 can be applied as a post-exposure therapy to modulate the locked-in immune response and thereby facilitate an effective immune response to virus variants. Thus, a deceptive, locked-in immune response to HIV antigens is modified by applying 1F7 as therapeutic vaccine in chronically HIV-infected individuals and AIDS patients in order to achieve an immune response able to overcome virus variants that had escaped the previous, locked-in immune response set by HIV infection. (Kohler, H., et al, Immunology Today (1994)]. Deceptive imprinting has been described in a review as an immune reaction in HIV and parasitic infections, i.e., it is based on xe2x80x9cOriginal Antigenic Sinxe2x80x9d (OAS) of an immune response. [Veljkovic, V., et al., Vaccine (2001), in press]. OAS is defined for infectious pathogenic organisms such as influenza, dengue, malaria, and HIV, as imprinting or B cell dominance of the host""s immune response by the antigenic make-up of the virus or parasite at first encountered during infection. The imprinting leads to an insufficient or xe2x80x9cdeceptivexe2x80x9d immune response due to B cell dominance that prevents an adequate immune response to the challenge by a rapidly mutating virus or parasite within the host.
(2) 1F7 can be used as a prophylactic co-vaccine when applying gp120-based and other HIV protein-based prophylactic subunit vaccines in HIV seronegative individuals at risk of HIV infection. In this approach, deceptive imprinting by HIV potentially induced by subunit vaccines as recombinant gp120 (Veljkovic, et al., 2001) can be counteracted by co-administration of 1F7.
(3) 1F7 can be used during xe2x80x9cstructured therapy interruptionxe2x80x9d (STI) in HIV-infected patients treated early on with antiviral drugs. [Rosenberg, E., Nature (2000) 407: 523]. STI has been reported to restore a short-term immune response on T cell levels being able to fight virus variants. Co-application of the 1F7 antibody can be helpful to prolong a diversified immune response to HIV induced by STI.
In view of the demonstrated therapeutic potential of Mab 1F7 in a suitable primate model, the immunoreactive fragments of 1F7 can also be employed for therapeutic benefit. Thus, recombinant DNA techniques can be employed to isolate and manipulate the nucleotide sequences encoding the variable heavy (VH) and variable light (VL) regions of the 1F7 antibody. For instance, the variable chains of the murine antibody can be fused to the constant gamma or kappa/lambda regions of human immunoglobulins (Igs) to afford human/murine chimeras, which display the complementarity-determining regions (CDRs) and/or framework-determining regions (FRs) of 1F7. Such human/murine chimeric antibodies are expected to reduce the human anti-murine antibody (HAMA) response often encountered in passive immunization therapy, thereby affording better-tolerated vaccines. In general, such chimeric antibodies show an increase in biological half-life in vivo. They also are capable of efficiently mediating the antibody-dependent complement cascade (ADCC), antibody-dependent macrophage cytotoxicity (ADMC), and complement fixation.
Chimeric antibodies often, however, remain immunogenic in primates due to the presence of murine variable regions. Another approach to reducing the immunogenicity of murine variable regions is to mutate the VH and VL chains of the 1F7 antibody to homologous human sequences, while retaining the murine CDRs and/or FRs, i.e., the variable chains are xe2x80x9chumanizedxe2x80x9d [Mateo, C, et al., Immunotech., (1997), 3: 71-81]. Linking the modified murine variable regions to human constant regions can afford chimeric antibodies in which the HAMA response is nullified.
The development of humanized murine antibodies has afforded exciting new therapeutics in recent years for a variety of illnesses, notably, non-Hodgkin""s lymphoma, breast cancer, among others. Antibody products now account for the single largest group of biotechnology-derived molecules in clinical trials; to date, however, no antibody product has been approved for the treatment of HIV infection.
The present invention is directed to one or more isolated polynucleotides containing at least one nucleotide sequence encoding a complementarity-determining region (CDR) and/or a framework-determining region (FR) of an anti-idiotypic antibody that binds to the idiotopes of anti-HIV-1 antibodies. The aforementioned anti-idiotypic antibody is preferably Mab 1F7 produced by the hybridoma having American Type Culture Collection (ATCC) Accession No. HB 11286. Depending upon the precise nature of the polynucleotide, a single CDR, a single FR, combinations of these, or an entire variable heavy (VH) or variable light (VL) chain of the anti-idiotypic antibody can be encoded. Moreover, murine regions outside the CDRs can be mutated or replaced with human immunoglobulin sequences in order to humanize the antibody or fragment thereof.
Typically, a DNA molecule of the invention is used to obtain a polypeptide containing more than one CDR and/or FR of the 1F7 antibody. For instance, the DNA molecule can be used to transform a suitable non-human host to express a VH and/or VL chain peptide of 1F7. The VH and VL chain peptide can be fused to the corresponding constant heavy and light chain peptides for mice or humans by covalently linking the DNA molecules to the appropriate segments of the immunoglobulin genes. Whenever the complete heavy and light chains are co-expressed, an assembled murine 1F7 or human/murine chimeric analog of 1F7 is provided, which can be used in a passive immunization protocol. Alternatively, a DNA molecule encoding the at least one CDR and/or FR of the 1F7 antibody can be used directly as a DNA vaccine, e.g., by injection of muscle cells with xe2x80x9cnaked DNAxe2x80x9d. The afforded antibody product is thus: (i) a xe2x80x9chumanizedxe2x80x9d antibody; (ii) a CDR/FR grafted antibody; or (iii) a xe2x80x9cde-immunizedxe2x80x9d antibody with removed or altered murine antigenic residues. The therapeutic effect of such an antibody is a broadening and increase of virus neutralization.
A particularly preferred aspect of the invention employs murine nucleotide sequences encoding the CDRs of the VH and VL chains of Mab 1F7, which have the amino acid sequences shown in SEQ ID NOS: 11, 15, 19, 28, 32 and 36. Also preferred are those murine nucleotide sequences encoding the FRs of the VH and VL chains of 1F7 having the amino acid sequences shown in SEQ ID NOS: 9, 13, 17, 21, 26, 30, 34 and 38. It is to be appreciated that the present invention contemplates and includes all nucleotide sequences equivalently encoding the aforementioned murine amino acid sequences by virtue of the degeneracy of the genetic code.